Use of a warmer for promoting a biological reaction

ABSTRACT

The present invention relates to the use of warmers, or autonomous heat packs, for heating and maintaining a solution on at a suitable temperature, for the period of time required to accomplish a chemical, biochemical or biological reaction, in particular in molecular biology or cell biology applications. Biology kits containing warmers are also part of this invention.

The present invention relates to the field of molecular and cell biologykits, and also to that of kits for carrying out a chemical reactionrequiring a moderate provision of heat. The subject of the presentinvention is in particular kits comprising a simple and inexpensiveautonomous heating means for treating biopsies.

For several years, the grafting of cells, in particular of autologouscells, has been essential as an effective and safe means for promotingthe regeneration of diseased or damaged tissues. Certain therapeuticprotocols require the proliferation and/or the modification of the cellsex vivo before reimplantation. Other protocols essentially provide forthe sampling of cells from a healthy part of the tissue concerned andthe virtually immediate reimplantation thereof at the lesion to betreated. This is in particular the case with protocols for the treatmentof skin diseases or lesions such as certain burns (the extent of whichdoes not require cell expansion), post-traumatic and post-operativehypochromia, achromatic dermatosis, scars, etc. In these protocols, thetreatment of the samples essentially amounts to a more or less thoroughdissociation of the cells, followed, where appropriate, by theseparation of various cell types in order to select the cellsappropriate for the intended application (melanocytes for achromaticdermatosis, for example) and by filtration in order to remove theaggregates. The cell dissociation is usually carried out by incubatingthe sample in a trypsin solution. The optimal temperature for trypsinactivity is 37° C. At this temperature, and depending on theconcentration of enzyme and the desired degree of dissociation, theincubation times described in the literature are between 50 minutes and3 hours (Guerra et al., 2003; Mulekar, 2003; van Geel et al., 2004). Ata lower temperature, the enzyme is less active, and it is necessary toprolong the incubation time by several hours, thereby making itimpossible to carry out all the steps of the protocol in the same day(Gauthier and Surleve-Bazeille, 1992). For this reason, these protocolsare currently carried out at least partially in structures which havetechnical platforms comprising an incubator, which is not often the casewith dermatology practices.

In order to enable practitioners not equipped with an incubator to treatpatients suffering from vitiligo, the company Clinical Cell Cultureprovides a kit (ReCell®) comprising the consumables required forcarrying out all the steps, from the sampling to the reimplantation ofthe cells, and also an electronic heating unit equipped with batteries.This device has two major drawbacks, which are its very high price andits ecological impact.

The present invention provides an advantageous alternative to the priorart solutions, in particular to the ReCell® kit, since it is based onthe use of warmers for heating and maintaining an enzymatic solution ata suitable temperature, during the period of time required for theaction of the enzyme. The term “warmer” denotes herein the small objectscapable of emitting heat, also called “magical heat packs” or“autonomous heat packs”, the operation of which is based on exothermicphysical or chemical processes. They are commonly used to heat the handsor feet exposed to cold. In the rest of the text, the terms “warmer” and“heat pack” are used without distinction. By way of example of a warmerbased on a physical process, mention may be made of the reusable warmersconsisting of a pouch containing a supercooled sodium acetate-saturatedaqueous solution. By bending a metal disk inside the liquid, crystals ofsolidified acetate are generated, which trigger crystallization, and thesolution becomes solid. Since this phase transition takes place at themelting point (54° for a 20% solution, for example), there is heating ofthe pouch and then cooling to ambient temperature once thesolidification is complete. When the pouch is cooled, it is possible toreturn the sodium acetate (which has become solid) to the liquid state,by placing the pouch in very hot water. Chemical warmers also exist, theconstituent of which is activated by oxidation on contact with air. Theyare effective for longer (from 8 to 60 hours compared with approximately1 hour for supercooled sodium acetate-based warmers) but can only beused once.

The inventors have shown (examples 1 and 3 below) that a solution placedin a container, typically a Petri dish, placed on a warmer, reaches, ina few minutes, an optimum temperature for the activity of numerousenzymes, such as trypsin, and stays at said temperature for at least 15to 20 minutes.

The invention therefore relates, firstly, to a process for carrying outa biological, biochemical or chemical reaction requiring an incubationat a temperature between 30 and 40° C., characterized in that itcomprises a step of activating a warmer, the operation of which is basedon an exothermic physical or chemical process, and a step of bringingsaid warmer into contact with a container containing the reagentsinvolved in said reaction. This process is particularly advantageous inthe context of a biological, medical or diagnostic application.

In one particular implementation of this process, the step of bringingthe warmer into contact with the container is carried out by placing thewarmer and at least the lower part of the container in a cavity of asupport provided for this purpose. Such a support makes it possible,firstly, to properly wedge the container on the warmer, thus limitingthe risks of tipping. In addition, the use of an insulating support,having a low thermal conductivity, makes it possible to limit heatlosses from the warmer and to promote transfer of the heat from thewarmer to the solution to be heated. In one preferred implementation,the depth of the cavity makes it possible to place therein the warmerand only the lower part of the container. In this way, the container isproperly wedged on the warmer, there is no risk of it sliding, and itremains easy for the operator to grasp.

In the above processes, the use of a warmer and, where appropriate, aninsulating support, makes it possible to maintain the temperature of abiological, biochemical or chemical solution between 30 and 40° C. forthe required period of time (from a few minutes to a few tens ofminutes, for example 5, 10, 15, 18, 20 minutes or more). This use of thewarmer, which is a simple, inexpensive object generally intended foroutdoor leisure activities (skiing, mountaineering, etc.), for carryingout a biological (molecular or cell biology) or chemical process as areplacement for a sophisticated piece of laboratory equipment, is asadvantageous as it is surprising, since it enables considerable savingswithout impairing the quality of the results obtained.

In the aforementioned, the term “biological reaction” is applied to anyreaction involving elements derived from a living being, for instanceliving cells (animal cells originating from a biopsy, plant cells,yeasts or bacteria), viruses, organelles, enzymes, metabolic products,etc. A “biochemical reaction” uses substances involved in chemicalreactions of living matter (enzymes, sugars, lipids, etc.).

According to one particular embodiment of the process of the invention,the container contains an enzymatic solution and the contact between thewarmer and the container is maintained for at least 10 minutes.According to one particular embodiment, the warmer is used in thecontext of a cell biology process, and the enzymatic solution containscells originating from a biopsy, for example a sample of tissue to bedissociated. In this case, the enzymatic solution contains, for example,trypsin.

For reactions requiring an incubation of less than one hour, the warmerpreferably consists of a hermetic plastic pouch containing a sodiumacetate-saturated aqueous solution. However, the invention can also beused with a chemical warmer, enabling a longer incubation.

According to one particular embodiment of the process of the invention,the warmer used, of pouch type containing sodium acetate, is a disk ofwhich the thickness is between 3 and 7 mm, preferably 4 to 6 mm, and thediameter is between 7 and 11 cm, preferably 8 to 10 cm, and the solutioncontaining the reagents involved in the reaction has a volume between 3and 10 ml and is contained in a Petri dish having a diameter between 7and 11 cm, preferably between 8 and 10 cm, or a compartment of saiddish. The term “Petri dish” is intended to mean herein a shallowtransparent cylindrical dish, made of glass or plastic, with a lid. Incertain particular implementations of the invention, the Petri dish iscompartmentalized, for example by means of a small wall along onediameter. When a support is used in the context of this particularembodiment, the cavity intended to receive the warmer is also circular,with a diameter slightly larger than that of the warmer (between 7 and11.5 cm), and has a depth of between 5 and 20 mm.

The present invention also relates to a process for dissociating cellsderived from a freshly taken tissue sample, comprising the followingsteps:

-   (i) initiating the crystallization in a warmer consisting of a    hermetic plastic pouch containing a sodium acetate-saturated aqueous    solution; optionally, placing said warmer in a cavity provided for    this purpose in a support, preferably an insulating support;-   (ii) placing on said warmer a container containing a solution of    trypsin having a concentration between 0.2% and 1%;-   (iii) placing the tissue sample in said solution and leaving to    incubate for at least 10 minutes, preferably 15 to 20 minutes;-   (iv) rinsing the tissue sample.

Of course, during the implementation of this process, care will be takento ensure that the geometries of the warmer, of the container and, whereappropriate, of the cavity of the insulating support are such that thereis a large surface area of exchange between the warmer and thecontainer. According to one particular embodiment, the warmer has a diskshape and the container is a Petri dish, as mentioned above. In thiscase, the diameters of these two elements are preferably identical orvirtually identical; if a support is used, the cavity of the supportintended to receive at least the warmer is also circular, with adiameter slightly larger than the diameter of the warmer and of thecontainer.

In the process above, step (iv) can, where appropriate, be preceded orfollowed by a step of inhibiting the trypsin, by adding an inhibitor.However, according to one preferred embodiment of the process accordingto the invention, no trypsin inhibitor is added, the rinsing of thetissue sample being sufficient to stop the action of the trypsin. Thismakes it possible in particular to limit the number of substances thatwill be applied to the patient with the cells.

The present invention also relates to a process for preparing a cellsuspension suitable for application to the skin of a patient, comprisingthe following steps:

-   (i) carrying out the dissociation process described above on a skin    tissue sample comprising cells appropriate for a graft to a patient;-   (ii) harvesting the appropriate cells originating from the skin    sample and suspending them in a solution; where appropriate, this    step requires an intermediate step of separating and/or sorting    certain cells from the sample; and-   (iii) filtering the solution obtained in step (ii) on a cell sieve.

According to one particular embodiment, the process above comprises anadditional step of adding hyaluronic acid to the cell suspensionobtained in step (iii). This step makes it possible to obtain a viscousmixture which is easily applied in the wound bed. In addition, thehyaluronic acid promotes cell viability.

One particular application of the processes above is the treatment ofvitiligo; in this application, the cells recovered in step (ii) compriseat least melanocytes.

The present invention also relates to a kit for biological application(cell biology kit and/or molecular biology kit), characterized in thatit comprises a warmer, the operation of which is based on an exothermicphysical or chemical process.

According to one preferred embodiment of the kits according to thepresent invention, the warmer consists of a hermetic plastic pouchcontaining a supercooled sodium acetate-saturated aqueous solution.

According to one preferred embodiment of the kit of the presentinvention, the kit also contains a support which comprises a cavityintended to receive the warmer. This support comprises, for example, aplate of thermally insulating material having a thickness between 5 and35 mm, in which the cavity intended to receive the warmer has a depthbetween 5 and 25 mm. The support can also consist of a thinner plate(for example 1 mm thick), bent or assembled to form a hollow support.Preferably, the material used for the support has a thermal conductivityλ less than or equal to 0.4 W·m⁻¹·K⁻¹ at 20° C. More preferably, itsthermal conductivity is less than 0.15 W·m⁻¹·K⁻¹, or even less than 0.08W·m⁻¹·K⁻¹ or even less than or equal to 0.04 W·m⁻¹·K⁻¹. By way ofnonlimiting examples of usable materials, mention may be made of wood (λof plywood=0.11 W·m⁻¹·K⁻¹), cardboard (λ=0.07 W·m⁻¹·K⁻¹), cork (λ=0.04W·m⁻¹·K⁻¹), PVC (λ=0.2 W·m⁻¹·K⁻¹), polypropylene (λ=0.1 to 0.22W·m⁻¹·K⁻¹), rigid polyurethane foam (λ=0.025) and expanded polystyrene(λ=0.036). According to the applications envisioned for the kit, theinsulating nature of the support is more or less important. For example,for a kit intended to prepare, in a dermatology practice, a cellsuspension by means of a process as described above, the insulatingnature of the support is secondary, since the premises in which the kitwill be used are generally at at least 18° C., and the incubation timeof the cell sample with the trypsin is not very long. On the other hand,certain applications may impose extreme heat conditions and/or require alonger incubation time at a temperature between 30 and 40° c. In thiscase, it is important to use an insulating support, which can, whereappropriate, be supplemented with a lid for keeping the heat in thecontainer for longer. By way of nonlimiting examples of suchapplications requiring an incubation of more than one hour between 30and 40° C., mention may be made of the enzymatic digestion of cartilage,in order to isolate chondrocytes; the digestion of a testicular biopsy,in order to isolate germ cells; the obtaining of islets of Langerhansfrom a pancreatic biopsy; the digestion of dermis, in order to isolatefibroblasts; and the digestion of a gingival biopsy, in order to isolategingival fibroblasts.

The kits of the invention can also comprise a container, the base ofwhich has the same geometry as that of the warmer and, whereappropriate, of the cavity of the support, in order to allow efficientheat exchanges. For example, the warmer can be a disk of which thethickness is between 3 and 7 mm, preferably 4 to 6 mm, and the diameteris between 7 and 11 cm, preferably from 8 to 10 cm; in this case, thecontainer is preferably a Petri dish having a diameter approximatelyequal to that of the warmer and, if a support is present, the cavityintended to receive the warmer is also circular, with a diameterslightly larger than that of the warmer.

A kit according to the present invention can also contain an enzyme, forexample trypsin. Such a kit is, for example, designed for the treatmentof a biopsy, and comprises in particular the means required fordissociating the cells of said biopsy. According to one particularembodiment, this kit comprises a Petri dish divided into compartments,the diameter of which is identical or very close to that of the warmer,a cell sieve, and trypsin (in lyophilized form or in solution).

The following examples and figures illustrate the invention without,however, limiting its scope.

FIGURE LEGENDS

FIG. 1: Change in the temperature of a buffer solution in a Petri dish 9cm in diameter, placed on a sodium acetate warmer, having the shape of adisk with a diameter approximately equal to that of the Petri dish, overa period of 40 minutes, in a room at 20° C.

FIG. 2: Photos of the device comprising a support and a heat pack(warmer). 1: heat pack; 2: support; 3: compartmentalized dish placed onthe heat pack.

FIG. 3: Change in the temperature as a function of time, withoutsupport.

FIG. 4: Change in the temperature as a function of time, with thesupport V2.

FIG. 5: Change in the temperature as a function of time, with thesupport V3.

FIG. 6: Change in the temperature as a function of time (comparison ofthe means without support and with supports V2 or V3).

EXAMPLES Example 1 Temperature of the Medium in a Petri Dish Placed on aWarmer

The temperature of a (buffer) solution in a Petri dish 9 cm in diameter,placed on a sodium acetate warmer, having the shape of a disk with adiameter approximately equal to that of the Petri dish and a thicknessbetween 4 and 6 mm, was measured.

The results (FIG. 1) show quite a rapid increase in temperature, sincethe temperature reaches 30° C. in a few minutes. The temperature thenremains between 30 and 40° C. for a little more than 20 minutes.

These results showed good reproducibility.

Example 2 Application to Dermo-Epidermal Separation, in the Context ofthe Treatment of Vitiligo by Autologous Graft of Melanocytes

Using a thin skin biopsy (0.2 mm-0.3 mm), the bed of a wound is seededwith autologous cells.

The cell suspension obtained after disaggregation of the graft consistsof a mixed population, mainly basal keratinocyte cells, but alsoLangerhans cells, melanocytes and fibroblasts.

Materials Used

A warmer as described in example 1 and a disposable kit composed ofancillary elements and instruments: enzymatic and application solutions,sterile instruments, including a two-compartment Petri dish.

Protocol

A sterile surgical field is prepared.

Upon reception, the kit is stored at +4° C. until use. The kit is placedat ambient temperature 10 min before use.

Preparation and Heating of the Enzymatic Solution

-   -   Using a 5 ml syringe and a needle, take 5 ml of PBS solution and        inject them into the bottle of lyophilized trypsin. Homogenize        thoroughly before taking up the regenerated solution and in one        compartment of the two-compartment Petri dish.    -   Activate the heat pack instantaneously by crumpling the metal        plate; the crystal solidifies instantaneously.    -   Place the two-compartment Petri dish on the activated heat pack        and wait 5 min.

Taking a Skin Sample

The zone from which a sample is to be taken is delimited with a surgicalfelt tip pen, disinfected with 0.5% chlorhexidine in alcoholic solution,and anesthetized with 2% xylocaine. A thin strip of skin which has aminimum surface area of 4 cm² and a thickness of 0.2-0.3 mm is takenwith a dermatome.

Carrying Out the Dermo-Epidermal Separation (DES)

-   -   Using sterile forceps (not provided), transfer the biopsy into        the Petri dish compartment containing the trypsin in solution at        0.4%.    -   Incubate for 15 minutes.    -   Rinsing of the biopsy:        -   With the same 5 ml syringe and a needle, take 5 ml of PBS            solution and place in the second compartment of the Petri            dish.        -   At the end of the incubation, using sterile forceps,            transfer the biopsy into the second compartment, for rapid            rinsing with PBS.    -   Using sterile forceps, place the biopsy inside the lid of the        Petri dish, taking care to keep the direction of the        dermo-epidermal junction facing upward.    -   Using the forceps, separate the two fragments.

Preparation of the Cell Suspension

-   -   Using the second 5 ml syringe and a needle, take 1.5 ml of PBS        and place them on the biopsy fragments, scrape the cells from        the junction surfaces with a scalpel (not provided) and coarsely        cut up the epidermal part so as to produce a mixture of cells.    -   Set aside and immobilize the dermal part.    -   Tilt the Petri dish so as to suction up the entire volume using        the 5 ml syringe, draw the cells up into the syringe and suction        several times so as to create a cell suspension.

Cell Filtration

-   -   Transfer the cell suspension into the sieve that will have been        placed on the pot with the red cap.

Preparation of the Cell Suspension in Hyaluronic Acid

-   -   Remove the sieve.    -   Place the hyaluronic acid contained in the syringe (i.e. 1.5 ml)        in the pot with the red cap containing the filtered cell        suspension.    -   Using the syringe, mix the hyaluronic acid and the cell        suspension so as to obtain a viscous but homogeneous suspension.    -   The suspension thus prepared is ready to be deposited by syringe        on the wound bed.

Results

The protocol above was carried out several times, on three differentskin samples, using two trypsin concentrations (0.4% and 0.8%).

The results obtained are summarized in table 1 below.

TABLE 1 Number of Presence of Trypsin cells iso- Cell Cloningmelanocytes Identi- concen- lated ×10⁶/ viability efficiency by flowfication tration cm² (%) test cytometry Biopsy 1 0.4% 1.2 95.0%3.4% >0.1% 0.8% 1.2 97.0% 3.4% >0.1% Biopsy 2 0.4% 3.4 98.0% 3.3% >0.1%0.8% 2.3 97.0% 3.8% >0.1% Biopsy 3 0.4% 4.2 97.4% 4.6% >0.1% 0.8% 3.999.4% 4.5% >0.1%

Trypsin Concentration

The process for obtaining the active substance was adapted from thetechnique described by van Geel et al. (van Geel et al., 2004).

The concentration of the trypsin solution was optimized (0.4% or 0.8%trypsin instead of 0.25% trypsin/0.08% EDTA). Unlike the techniquedescribed by van Geel et al., the inhibition of the trypsin was notcarried out by adding inhibitor of fetal calf serum type, but by rinsingwith PBS, in order to reduce the number of products of biological originused for the production of the product. The cell viability after thismode of trypsin inhibition was validated.

Cell Viability

The cell viability was determined according to a conventional cellculture technique: the trypan blue exclusion test. A volume-for-volumedilution of trypan blue and of the cell suspension was carried out in ahemolysis tube. After a contact time of 1 to 2 minutes, the mixture wasdeposited with a micropipette between slide and coverslip on a countingcell. The dead cells, stained blue, and the live cells, unstained, werecounted using a phase contrast optical microscope.

The cell viability was very satisfactory after isolation of theepidermal cells (greater than 90%).

The behavior of the cells after being put back in culture and thecloning efficiency were uniform from one biopsy to the other and showedthat the cell suspension contained cells capable of proliferating aftertrypsinization.

Number of Cells Isolated/cm² of Biopsy

Calculation carried out on the basis of the results of the testdescribed above.

The number of cells isolated was variable from one individual to anotherdepending on the size of the biopsies treated, but the cell yield/cm²was relatively constant. The cell density of the final suspensiontherefore depends greatly on the size of the biopsy treated.

Cloning Efficiency Test (CFE)

The cloning efficiency percentage makes it possible to evaluate theamount of cells capable of adhering and of forming colonies. A knownamount of epidermal cells was seeded into 3 T25 cm² flasks. Around the14th day of culture, when the clones were sufficiently large but notjoining up, the flasks were stained with a solution of crystal violet(10% formaldehyde/0.5% crystal violet, qs distilled water). The cloningefficiency was calculated by producing the ratio of the mean of thetotal number of colonies per T25 cm² flask×100 to the number of cellsseeded per T25 cm².

The behavior of the cells after being placed back in culture and thecloning efficiency were uniform from one biopsy to the other and showedthat the cell suspension contained cells capable of proliferating aftertrypsinization.

Melanocyte Content in the Cell Suspension by Flow Cytometry

The cell membrane was permeabilized in a solution of PBS-1% BSA (bovineserum albumin)-0.5% triton. The primary antibody specific for normalmelanocytes (NKI/beteb antibody) was incubated, and then rinsed withPBS-1% BSA. The secondary antibody (Alexa Fluor 488 donkey antimouseantibody) was incubated and then rinsed with PBS-1% BSA. The labeledcells were taken up in PBS and passed through a FACS-SCAN.

The melanocyte/keratinocyte ratios in the epidermal suspensions obtainedwere comparable to those calculated by Guerra et al. (between 1:30 and1:200) (Guerra et al., 2003).

In the process described here, the isolated epidermal cells were notplaced in culture and the entire process was carried out in one day(biopsy, production of the final product and grafting of nonculturedautologous epidermal cells).

Example 3 Use of an Insulating Support for Optimizing the Increasing andthe Maintaining of the Temperature of the Medium Using the Warmer 3.1.Materials and Methods Materials and Reagents

-   -   PVC heat packs (warmers), EC labeling.    -   Support plates consisting of a polypropylene (PP) sheet 1 to 2        mm thick, folded to obtain a support with a total thickness of        15 mm, having a circular cavity 90 mm in diameter (FIG. 2).    -   Compartmentalized Petri dish (ref. Dutscher 020012).    -   Temperature probe (thermobouton) programmed to operate with the        ThermoTrack V4 and/or A BT 529 software.

Methods

Six milliliters of PBS stored at +4° C., equilibrated at ambienttemperature for 20 minutes, are placed in one of the compartments of aPetri dish 90 mm in diameter, in which a thermobouton probe (device forrecording temperature variations) is placed in order to take temperaturerecordings every minute. The Petri dish is then covered with its lid.

The thermobouton probe is in contact with the medium, making it possibleto measure temperature variations directly within the solution, afteractivation of the heat pack. The experiment is carried out by placing ornot placing the heat pack and the Petri dish in the circular cavity ofthe support (FIG. 2). Three different supports were tested. They differin terms of the more or less central position of the circular cavityintended to receive the warmer and the Petri dish.

Each condition was tested 5 to 6 times, with different temperatureprobes. The temperature measurements were carried out for 20 min. Themean of the temperatures was calculated over all the values recordedduring this period of time.

A statistical analysis of the temperatures measured in the medium withor without support was carried out by means of a student's t test.

3.2. Results

Change in the Temperature of the Medium with a Heat Pack without Support

The temperature data collected by the temperature probe immersed in themedium are represented diagrammatically in FIG. 3. The mean temperatureover the 6 tests was 35.5° C., with a mean standard deviation of 3.9° C.

These results demonstrate the reproducibility of the temperaturesobtained with the various warmers, without support.

Change in the Temperature of the Medium with a Heat Pack in a Support

Two supports (prototypes V2 and V3) were tested. They differ in terms ofthe position of the circular cavity intended to receive the warmer.Indeed, in the support V3, this cavity was centralized so as to allowthe heat inside the support to be uniform, in order to optimize theincreasing and the maintaining of the temperature in thecompartmentalized Petri dish placed on the warmer.

The temperature data collected for 20 minutes by the temperature probeimmersed in the medium are represented diagrammatically in the graphspresented in FIG. 4 (support V2), in FIG. 5 (support V3) and in FIG. 6(comparison of the two supports and of the temperatures obtained withoutsupport), and summarized in the table below.

TABLE 2 Period of time during which temper- Time to Mean ature main-Maximum reach the temperature tained >35° temperature maximum of the 6C.* reached temperature tests Test 18 min 39.5° C. 9 min 35.5° C. ±without 3.9° C. support Test with 18 min 40.5° C. 6 min 36.7° C. ±support V2 3.6° C. Test with 18 min   40° C. 9 min 36.3° C. ± support V31° C. *period of time obtained over a total time of the experiment of 21minutes

These results demonstrate:

-   -   the reproducibility between the warmers;    -   the temperatures reached are higher when the warmers are placed        in a support (1.1° C. of difference with V2 and 0.8° C. with        V3);    -   with or without support, the medium contained in the        compartmentalized Petri dish is maintained at a temperature        above 35° C. for at least 18 minutes;    -   without support, the maximum temperature of 39.5° C. is reached        in 9 min, whereas, with the support V2, it reaches 40.5° C. in 6        min.

In order to determine whether the differences observed are statisticallysignificant, a statistical analysis by means of a student's t test wascarried out using the values obtained with the support V3. This testindicates a probability of 0.03<0.05, demonstrating that the differencein the temperatures obtained without support and with the support V3 issignificant (table 3). The support V3 therefore makes it possible toobtain a temperature which is on average higher than without support,more rapidly and more reproducibly (s.d.±1° C.)

TABLE 3 Mean without Mean with support support 35.4 35.9 35.9 35.7 35.736.9 35.3 36.3 35.3 36.8 35.6 NA Mean 35.5 36.32 t 0.03

The supports therefore make it possible to optimize the increase intemperature and/or the maintaining thereof.

The use of the support of the VitiCell® kit during an enzymaticdigestion of a thin skin biopsy in order to produce a dermo-epidermalseparation thereof, according to the process for producing epidermalsuspensions described in example 2, makes it possible to obtain onaverage a higher temperature and more rapidly. The support therefore hasa double advantage: work plate and optimization of the temperatureincrease of the heat packs.

REFERENCES

-   Gauthier, Y. and Surleve-Bazeille, J. E. (1992) Autologous grafting    with noncultured melanocytes: a simplified method for treatment of    depigmented lesions. J Am Acad Dermatol, 26, 191-194.-   Guerra, L., Primavera, G., Raskovic, D., Pellegrini, G., Golisano,    O., Bondanza, S., Paterna, P., Sonego, G., Gobello, T., Atzori, F.,    Piazza, P., Luci, A. and De Luca, M. (2003) Erbium:YAG laser and    cultured epidermis in the surgical therapy of stable vitiligo. Arch    Dermatol, 139, 1303-1310.-   Mulekar, S. V. (2003) Melanocyte-keratinocyte cell transplantation    for stable vitiligo. Int J Dermatol, 42, 132-136.-   van Geel, N., Ongenae, K., De Mil, M., Haeghen, Y. V., Vervaet, C.    and Naeyaert, J. M. (2004) Double-blind placebo-controlled study of    autologous transplanted epidermal cell suspensions for repigmenting    vitiligo. Arch Dermatol, 140, 1203-1208.

1. A process for carrying out a biological, biochemical or chemicalreaction requiring an incubation at a temperature between 30 and 40° C.,the process comprising a step of activating a warmer, the operation ofwhich is based on an exothermic physical or chemical process, and a stepof bringing said warmer into contact with a container containing thereagents involved in said reaction.
 2. The process as claimed in claim1, wherein the step of bringing the warmer into contact with thecontainer is carried out by placing the warmer and at least the lowerpart of the container in a cavity of a support provided for thispurpose.
 3. The process as claimed in claim 1, wherein the containercontains an enzymatic solution and in that the contact between thewarmer and the container is maintained for at least 10 minutes.
 4. Theprocess as claimed in claim 1, wherein the warmer consists of a hermeticplastic pouch containing a sodium acetate-saturated aqueous solution. 5.The process as claimed in claim 4, wherein the volume of the solutioncontaining the reagents involved in the reaction is between 3 and 10 ml,the container containing said solution is a Petri dish having a diameterbetween 7 and 11 cm or a compartment of said dish and in that the warmeris a disk of which the thickness is between 3 and 7 mm and the diameteris between 7 and 11 cm.
 6. The process as claimed in claim 2, whereinthe cavity of the support intended to receive the warmer is circular,with a diameter between 7 and 11.5 cm, and has a depth between 5 and 20mm.
 7. The process as claimed in claim 1, for dissociating cells derivedfrom a tissue sample, comprising the following steps: (i) initiating thecrystallization in the warmer consisting of a hermetic plastic pouchcontaining a sodium acetate-saturated aqueous solution; (ii) placing onsaid warmer the container containing a solution of trypsin having aconcentration between 0.2% and 1%; (iii) placing the tissue sample insaid solution and leaving to incubate for at least 10 minutes; and (iv)rinsing the tissue sample.
 8. The process as claimed in claim 7, forpreparing a cell suspension appropriate for application to the skin of apatient, comprising the following steps: (i) subjecting a skin tissuesample comprising cells appropriate for a graft to a patient to theprocess as claimed in claim 7; (ii) harvesting the appropriate cellsoriginating from the skin sample and suspending them in a solution; and(iii) filtering the solution obtained in step (ii) on a cell sieve. 9.The process as claimed in claim 8, comprising an additional step ofadding hyaluronic acid to the cell suspension obtained in step (iii).10. The process as claimed in claim 8, for the treatment of vitiligo,wherein, in step (ii), the melanocytes are recovered.
 11. A kit forbiological application, characterized in that it comprises a warmer, theoperation of which is based on an exothermic physical or chemicalprocess.
 12. The kit as claimed in claim 11, wherein the warmer consistsof a hermetic plastic pouch containing a supercooled sodiumacetate-saturated aqueous solution.
 13. The kit as claimed in claim 11further comprising a support which has a cavity intended to receive saidwarmer.
 14. The kit as claimed in claim 11 further comprising acontainer, the base of which has the same geometry as that of thewarmer.
 15. The kit as claimed in claim 11 further comprising an enzyme.